1. Field of the Invention
This invention pertains to steroid derivatives containing an imidazole group wherein said derivatives are radioactively labeled. Said radioactively labeled derivatized steroids are useful in radioimmunoassay methods for assaying analogous, nonlabeled, underivatized steroids.
2. Description of the Prior Art
The use of .sup.125 I-tracers in steroid immunoassays has certain advantages in comparison with tritiated tracers. Besides the obvious advantage of the higher specific activity of the radioiodine over tritium, these .sup.125 I-tracers also result in a simpler and less expensive counting system (gamma counting as opposed to liquid scintillation counting). To produce .sup.125 I-labeles useful for steroid radioimmunoassay (RIA) techniques, two main approaches have been used. One approach entails the use of tyrosyl methyl ester (TME) derivatives of steroids which can be readily iodinated. U. Barbieri, A. Massaglia, M. Zannino, and U. Rosa, J. of Chromat., 69:151 (1972) and A. R. Midgley, G. D. Niswender, V. L. Gay, and L. E. Reichert, Recent Progr. Hormone, 27:325 (1971). A second method entails iodinating steroid-protein constituents that have been used to raise antisera and using said iodinated steroid-protein conjugates as tracers. A. R. Midgley et al., supra, and S. L. Jeffcoate, E. D. Gilby, and R. Edwards, Clinica Chemica Acta, 42:343 (1973). A Massaglia, U. Barbieri, and C. SiriUpathum, International J. of Applied Radiation and Isotopes, 24:455 (1973), reported on the synthesis, purification, and iodination of cortisol-21-hemisuccinyl TME and cortisol-3-(O-carboxymethyl) oxime-TME derivatives. In the same year, R. Mavano, C. Dotti, and P. Grosso, Clinica Chemica Acta, 47:167 (1973), reported on the employment of .sup.125 I-labeled cortisol-21-hemisuccinyl-TME as a tracer in the competitive protein binding assay using transcortin as the binder. Since iodination of an estradiol TME derivative results in iodine substitution of the A ring and consequently a loss of the immunoreactivity of the tracer (P. W. Nars and W. M. Hunter, J. Endocr., 57:XLVII (1973) and E. D. Gilby, S. L. Jeffcoate, and R. Edwards, J. Endocr., 58:XX (1973)), histamine was first iodinated and subsequently coupled to an estradiol-6-(O-carboxymethyl) oxime hapten using a mixed anhydride synthesis. B. F. Erlinger, F. Borek, F. M. Beiser, and S. Lieberman, J. Biol. Chem., 228:713 (1957). The same mixed anhydride synthesis procedure has also been used to prepare estradiol-6-(O-carboxymethyl) oxime-.sup.125 I-tyramine (P. Linberg and L. E. Edquist, Clinica Chemica Acta, 53:169 (1974)) and progesterone-3-(O-carboxymethyl) oxime .sup.125 I-histamine (J. J. Scarisbrick and E. H. D. Cameron, J. of Steroid Biochem., 6:51 (1975)) tracers for use in RIA.
It has been discovered that unlike tyrosine derivatives which yield a tracer possessing a lower affinity for the antisera than the unlabeled steroid and also giving a high nonspecific background count in polystyrene and polypropylene vials, particularly in the presence of naturally occurring serum constituents histamine derivatives of steroids alleviate these problems and therefore yield labeled haptens which are much superior for use in RIA.